Journal: Frontiers in Pharmacology

Article Title: Astragaloside IV Attenuates Podocyte Apoptosis Mediated by Endoplasmic Reticulum Stress through Upregulating Sarco/Endoplasmic Reticulum Ca 2+ -ATPase 2 Expression in Diabetic Nephropathy

doi: 10.3389/fphar.2016.00500

Figure Lengend Snippet: AS-IV attenuated ER stress and ER stress-induced apoptotic pathway in the kidney of db/db mice. (A,B) Representative pictures for GRP78 immunohistochemistry staining (A) and quantification of IOD (B) for GRP78 in each group. Scale bars, 10 μm; Original magnification, ×400. (C,D) Western blot analyses (C) and densitometric quantification (D) of GRP78 expression in the kidney cortex lysates from each group. (E,F) Representative immunoblots (E) and densitometric quantification (F) of ATF6, p-PERK, p-eIF2α, ATF4, CHOP expression in total lysates of kidney cortex from each group. (G,H) Representative immunoblots (G) and densitometric quantification (H) of p-IRE1α, spliced-XBP1, TRAF2, ASK1, and phospho-JNK Thr183/Tyr185 expression in total lysates of kidney cortex from each group. (I,J) Representative immunoblots (I) and densitometric quantification (J) of cleaved caspase 12, cleaved caspase 9 and cleaved caspase 3 expression in total lysates of kidney cortex from each group. Data are expressed as mean ± SEM. n = 6. ∗∗ P < 0.01; # P < 0.05 and ## P < 0.01. One-way ANOVA and Newman–Keuls multiple comparisons test (B,D,F,H,J) .

Article Snippet: The following primary antibodies were used: eIF2α, JNK, phospho-JNK Thr183/Tyr185 , AIF, cyclophilin D, and ANT from Santa Cruz Biotechnology (Santa Cruz, CA, USA), GAPDH, SERCA1, inositol-requiring enzyme 1α (IRE1α), TNF receptor associated factor 2 (TRAF2), PERK, phospho-PERK Thr980 , phospho-eIF2α Ser51 , activating transcription factor 4 (ATF4), caspase 12, caspase 9, caspase 3, CHOP, and cytochrome c from Cell Signaling Technology (Danvers, MA, USA), SERCA2, Podocin, Nephrin, GRP78, ATF6, XBP1, voltage dependent anion channel 1 (VDAC1), APAF1, and PPARγ from Abcam company (Cambridge, MA, USA), phospho-IRE1α Ser724 from Novus Biologicals, SERCA3 from Proteintech Group (Chicago, IL, USA), ASK1 from ProSci Incorporated (San Diego, CA, USA).

Techniques: Immunohistochemistry, Staining, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Astragaloside IV Attenuates Podocyte Apoptosis Mediated by Endoplasmic Reticulum Stress through Upregulating Sarco/Endoplasmic Reticulum Ca 2+ -ATPase 2 Expression in Diabetic Nephropathy

doi: 10.3389/fphar.2016.00500

Figure Lengend Snippet: AS-IV restored SERCA2 expression and inhibited ER stress-mediated apoptosis in palmitate-stimulated podocytes. Podocytes were pretreated with or without AS-IV at the indicated concentrations (20, 40, and 80 μM respectively) for 12 h followed by 250 μM palmitate exposure for 24h. (A) Representative flow cytometry results for podocytes under different cultural conditions. (B) Semiquantitative data showing percentage of apoptotic podocytes under different cultural conditions. (C,D) Representative immunoblots (C) and quantification (D) of Podocin, SERCA2 and GRP78 under different conditions. (E) SERCA activity under different conditions. (F,G) Representative immunoblots (F) and quantification (G) of key molecules in ATF6-CHOP/PERK–eIF-2α–ATF4–CHOP signaling pathways under different conditions. (H,I) Representative immunoblots (H) and quantification (I) of key molecules in IRE1–XBP1/IRE1-TRAF2–ASK1–JNK signaling pathway. (J,K) Representative immunoblots (J) and quantification (K) of cleaved caspase 12 and cleaved caspase 3 under different conditions. Data are expressed as mean ± SEM. n = 3–5. ∗∗ P < 0.01; # P < 0.05 and ## P < 0.01. One-way ANOVA and Newman–Keuls multiple comparisons test (B,D,E,G,I,K) .

Article Snippet: The following primary antibodies were used: eIF2α, JNK, phospho-JNK Thr183/Tyr185 , AIF, cyclophilin D, and ANT from Santa Cruz Biotechnology (Santa Cruz, CA, USA), GAPDH, SERCA1, inositol-requiring enzyme 1α (IRE1α), TNF receptor associated factor 2 (TRAF2), PERK, phospho-PERK Thr980 , phospho-eIF2α Ser51 , activating transcription factor 4 (ATF4), caspase 12, caspase 9, caspase 3, CHOP, and cytochrome c from Cell Signaling Technology (Danvers, MA, USA), SERCA2, Podocin, Nephrin, GRP78, ATF6, XBP1, voltage dependent anion channel 1 (VDAC1), APAF1, and PPARγ from Abcam company (Cambridge, MA, USA), phospho-IRE1α Ser724 from Novus Biologicals, SERCA3 from Proteintech Group (Chicago, IL, USA), ASK1 from ProSci Incorporated (San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Western Blot, Activity Assay, Protein-Protein interactions

Journal: Oncology Letters

Article Title: Effect of CXCR4 silencing with shRNA on MAPK signaling in ovarian cancer

doi: 10.3892/ol.2018.8550

Figure Lengend Snippet: Expression of ASK1 mRNA following C-X-C chemokine receptor type 4-short hairpin RNA transfection.

Article Snippet: Other reagents included the oligonucleotide probe for CXCR4 , pGenesil plasmid and Escherichia coli DH5α (Wuhan Genesil Biotechnology, Wuhan, China); a one-step plasmid extraction kit (Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China); TRIzol RNA kit [Tiangen Biotech (Beijing) Co., Ltd, Beijing, China]; Lipofectamine ® 2000 transfection kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); primers for apoptosis signal-regulating kinase 1 (ASK1) and GAPDH (Shanghai Sangong Pharmaceutical Co., Ltd.); rabbit anti-extracellular-signal-regulated kinase (ERK)1/2 (cat. no. 9102) and rabbit anti phosphorylated (p)-c-Jun (Ser 73 ) (cat. no. 9164s) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) (dilutions, 1:100); PrimeScript ® RT reagent kit (Perfect Real Time) and SYBR ® Premix Ex Taq™ II (Perfect Real Time) (Takara Biotechnology, Co., Ltd., Dalian, China); and a diaminobenzidine (DAB) kit (PV-9000 kit reagents 1 and 2; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Oncology Letters

Article Title: Effect of CXCR4 silencing with shRNA on MAPK signaling in ovarian cancer

doi: 10.3892/ol.2018.8550

Figure Lengend Snippet: Immunocytochemical determination of ERK1/2 protein expression at 48 h after transfection. (A) Blank control, (B) empty vector group, and (C) interference group). Magnification, ×100. Lane 1, 2 and 3 represent blank control, empty vector and interference groups, respectively, in the SW626 cell line. ERK1/2, extracellular-signal-regulated kinase 1/2.

Article Snippet: Other reagents included the oligonucleotide probe for CXCR4 , pGenesil plasmid and Escherichia coli DH5α (Wuhan Genesil Biotechnology, Wuhan, China); a one-step plasmid extraction kit (Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China); TRIzol RNA kit [Tiangen Biotech (Beijing) Co., Ltd, Beijing, China]; Lipofectamine ® 2000 transfection kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); primers for apoptosis signal-regulating kinase 1 (ASK1) and GAPDH (Shanghai Sangong Pharmaceutical Co., Ltd.); rabbit anti-extracellular-signal-regulated kinase (ERK)1/2 (cat. no. 9102) and rabbit anti phosphorylated (p)-c-Jun (Ser 73 ) (cat. no. 9164s) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) (dilutions, 1:100); PrimeScript ® RT reagent kit (Perfect Real Time) and SYBR ® Premix Ex Taq™ II (Perfect Real Time) (Takara Biotechnology, Co., Ltd., Dalian, China); and a diaminobenzidine (DAB) kit (PV-9000 kit reagents 1 and 2; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Oncology Letters

Article Title: Effect of CXCR4 silencing with shRNA on MAPK signaling in ovarian cancer

doi: 10.3892/ol.2018.8550

Figure Lengend Snippet: Western blot determination of ERK1/2 protein expression at 48 h after transfection (P<0.01 vs. the interference group). ERK1/2, extracellular-signal-regulated kinase 1/2. *P>0.05, empty vector vs. lank control; **P<0.001, interference vs. blank control and empty vector.

Article Snippet: Other reagents included the oligonucleotide probe for CXCR4 , pGenesil plasmid and Escherichia coli DH5α (Wuhan Genesil Biotechnology, Wuhan, China); a one-step plasmid extraction kit (Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China); TRIzol RNA kit [Tiangen Biotech (Beijing) Co., Ltd, Beijing, China]; Lipofectamine ® 2000 transfection kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); primers for apoptosis signal-regulating kinase 1 (ASK1) and GAPDH (Shanghai Sangong Pharmaceutical Co., Ltd.); rabbit anti-extracellular-signal-regulated kinase (ERK)1/2 (cat. no. 9102) and rabbit anti phosphorylated (p)-c-Jun (Ser 73 ) (cat. no. 9164s) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) (dilutions, 1:100); PrimeScript ® RT reagent kit (Perfect Real Time) and SYBR ® Premix Ex Taq™ II (Perfect Real Time) (Takara Biotechnology, Co., Ltd., Dalian, China); and a diaminobenzidine (DAB) kit (PV-9000 kit reagents 1 and 2; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation